Aflatoxins, which are produced by the fungi, Aspergillus spp., are highly toxic, naturally occurring compounds that infect staple crops like maize, wheat, sorghum, cassava, peanuts, coffee and cocoa, mostly in developing countries. The Partnership for Aflatoxin Control in Africa (PACA) reports that “aflatoxins cause between 5% and 30% of all liver cancer in the world, with the highest incidence of 40% occurring in Africa.”

Numerous organizations are working worldwide to reduce aflatoxin exposure by applying improved food safety measures. For those already exposed, finding a biomarker for early detection of aflatoxin-induced liver cancer could be the key to improved outcomes. That goal is at the center of the paper HAfTs are novel lncRNA transcripts from aflatoxin exposure, published in PLOS One in January 2018.

B. Alex Merrick, PhD, with the Division of the National Toxicology Program at the National Institute of Environmental Health Sciences, part of the National Institutes of Health, led the research in partnership with the David H. Murdock Research Institute (DHMRI). Previously, Merrick and DHMRI collaborated on a study of genomic and transcriptomic changes caused by heavy metal carcinogens in human prostate cells and the metagenomic regulators for the changes.

“Our team originally thought these new transcripts would be regular coding genes,” Merrick said, “but our data generated with DHMRI provided a more interesting finding”. Naming them hepatic aflatoxin transcripts, or Hafts, he found that most of the new transcripts were long non-coding RNAs (lncRNAs) involved in regulatory processes.

To discover longer sequences of Hafts, DHMRI performed RACE − the rapid amplification of cDNA ends − and Sanger sequencing. “RACE required a lot of troubleshooting to get a good quality amplicon and then the amplicons had to be cloned in bacteria and confirmed with sequencing,” explained Genomics Scientist Meredith Bostrom, PhD, a co-author on the paper.

By applying RACE, DHMRI Genomics scientists were able to provide complete transcripts of several novel RNA transcripts starting with only partial sequences. “We used the incomplete sequence data to perform a PCR to encompass the ends of the transcripts that we didn’t know,” Bostrom explained.

In the end, the DHMRI Genomics team developed a set of new protocols and provided valuable sequence information to help identify these new HAfT transcripts as lncRNAs for Merrick. “I really think this project shows our versatility,” she added. “A lot of our clients and collaborators are familiar with our quality sequencing and microarray data, but the RACE project is a little bit different. The technique involves a lot more troubleshooting and demonstrates our skills in adapting new protocols.”

In addition, Bostrom continued, “We are excited about the results. We hope that the increased understanding of the role of aflatoxin in the pathogenesis of the cancer leads to discovery of a biomarker for the disease or even a therapeutic target.”

Merrick added, “The next challenge will be to find out the functions of HAfTs and their possible human relevance.”